The Interaction Mechanisms Of Frankliniella Occidentalis(Pergande) And Phaseolus Vulgaris L.induced By Exogenous Ca

Frankliniella occidentalis（Pergande）,western flower thrips（WFT）,is a globally important pest,causing serious damage to commercial crops such as vegetables and ornamental flowers.Calcium（Ca）is an essential element for plants and is also a key regulator in plant response to abiotic and biological stimuli or stresses,participating in the transduction of many cascaded signals pathway in plants.In this dissertation,we studied the interactions between WFT and kidney bean plant induced by exogenous Ca.Firstly,the effects of different Ca Cl₂ concentrations on the resistance of kidney bean plant against WFT were studied and the most effective concentration was determined.Then,changes of feeding behavior,feeding damage area,feeding preference and life table parameters of WFT were compared in kidney bean plants treated with exogenous Ca and H₂O.Cytosolic calcium concentration([Ca²⁺]_cyt),calcium ion contents,jasmonic acid（JA）and salicylic acid（SA）contents,defense enzyme activities and m RNA expression levels on kidney bean plants with and without Ca Cl₂ treatment were determined after WFT infestation.Furthermore,Changes in the activities of protection enzymes,detoxification enzymes and m RNA transcription levels of WFT were determined when WFT feeding on Ca-treated plants and control plants.The defense responses of kidney bean plants induced by exogenous Ca and WFT feeding were analyzed,and anti-defense response of WFT were analyzed too.The interaction mechanisms of plant and WFT were revealed.The results will provide theoretical basis and practical guidance for integrated pest management on WFT by using exogenous Ca.The main results were as follows:1 Exogenous calcium induced the defense of kidney bean plant against WFTThe most effective concentration of Ca Cl₂ treatment was obtained via observing the developmental period and survival rate of WFT in kidney bean plant treated by different concentrations of Ca Cl₂ solution.Then,feeding behavior,feeding damage,feeding preference and life table parameters of WFT were further determined.The results showed that:（1）The developmental durations extended and survival rate decreased when WFT fed on kidney bean plant treated by different concentrations of exogenous Ca.The immature period was longest when WFT fed on 20 m M Ca Cl₂treatment and was significantly prolonged than control plant by 2.08 d.（2）Feeding behavior results showed that thrips made a significantly higher total number of P,NP and SP（mainly feeding period）in the Ca-treated plant than in the control plant,while thrips had significantly fewer LP（mainly feeding period）.The total duration of P and NP were significantly longer in the Ca Cl₂-treated plant than in the control plants,while the total duration of LP significantly decreased.（3）WFT feeding damage area significantly reduced compared to control plant no matter Ca Cl₂ treatment the adaxial or abaxial leaf of kidney bean plant.Compared with control leaves,the preference of WFT to adaxial or abaxial leaf of kidney bean plant treated by Ca Cl₂ significantly decreased,and feeding damage area also significantly reduced by 83.7%and 49.8%respectively.（4）The population parameters of intrinsic rate of increase（r）,finite rate of increase（λ）and net reproductive rate（R₀）of WFT fed on Ca-treated kidney bean plants were significantly lower than that of feeding on control plants,reduced by 15.8%,3.31%and 37.2%respectively.The mean generation time（T）remarkably increased by1.11 d.The results demonstrated that exogenous calcium could inducekidney bean plants against WFT.Exogenous Ca inducing systemic defense of kidney bean plant against WFT were investigated.Exogenous Ca was applied on the middle leaves of kidney bean plant,the effect on WFT feeding on the different parts of plant were different.（1）The period of immature WFT feeding on the middle leave was significant longer of 1.82 d than that of feeding the same position on control plant.WFT feeding on upper and lower leaves were significantly longer too,which extended by 0.87 d and 1.25 d respectively.The survival rate was significant difference on middle leaves between 2 thretments,and there were no significant differences in both the upperand lower leaves;（2）The total numbers of P,NP and SP of WFT on the middle leaves significantly increased than on the middle leaf of control plant,while the total number of LP significantly decreased.The total number of LP significantly decreased on the upper and middle leaves,and total number of NP significantly increased.Total number of NP of WFT significantly increased on the lower leaves only.The total duration of NP of WFT on the middle leave significantly increased than control plant,while the total duration of LP significantly decreased.Also,the total duration of SP on upper leaves significantly decreased.（3）The preference of WFT to the upper or lower leaves treated by Ca Cl₂significantly decreased than that on control plant,and the feeding damage area also significantly reduced by 53.3%and 51.2%respectively.（4）The number of WFT on Ca-treated kidney bean plant was significantly lower than on control plant,decreased by49.1%.These results indicated that exogenous calcium regulated the systemic defense of kidney bean plants against WFT.2 Exogenous calcium induced local and systemic defense of kidney bean plantThe Ca²⁺concentration or content,JA and SA content and the activities of defense enzymes in Ca-treated kidney bean plants infested by WFT were determined.（1）Wounding triggered rapidly and transiently increased in[Ca²⁺]_cyt in kidney bean leaves both treated by H₂O or Ca Cl₂,and then returned to control level after 3 min.In particular,[Ca²⁺]_cyt elevations in Ca-treated plant were significantly higher than those of control plant after wounding at 1 min and 2 min.（2）There was no significant difference in the Ca²⁺content between control and Ca-treated kidney bean plant without WFT infestation.When plants were exposed to WFT-infested,the Ca²⁺content in Ca-treated kidney bean plant significantly increased by 19.7%than control plants.（3）There were no significant differences in the contents of JA or SA in leaves of the plants treated with H₂O and Ca without WFT infestation.When subjected to WFT infestation for 24 h and 48 h,the JA content in Ca-treated kidney bean plant were significantly higher than that of control plants,while the content of SA significantly increased after infestation 6 h and 12 h.（4）There were no significant difference in the LOX,AOS,PAL and PR-2 activities between control and Ca-treated kidney bean plants without WFT infestation.However,when plants were subjected to WFT attack,the activities of LOX,AOS and PAL were much higher in Ca-treated plants than control plants,increased by 25.9%,47.9%,and26.3%respectively.There was no significant difference in PR-2 activity.The results showed that exogenous Ca induced resistance of kidney bean plant was dependent on the regulation of JA and SA signal transduction pathways.The Ca²⁺content,JA and SA contents and the activities of defense enzymes in leaves of different parts of kidney bean plant under Ca-treated the middle leaf were also determined.（1）Without the exposure to WFT-infested,Ca²⁺content was no significant difference in the middle leaves or upper leaves or lower leaves between control and Ca-treated kidney bean plants.When plants were exposed to WFT-infested,the Ca²⁺content in middle leaves of Ca-treated kidney bean plants was significantly higher than control plants,but the difference between the Ca-treated and control plants were not significant on upper and lower leaves（2）Without the exposure to WFT-infested,there were not significant difference in the contents of JA or SA in leaves of in different parts of kidney bean plant treated with H₂O and Ca Cl₂.When subjected to WFT infestation,the JA and SA contents showed an increasing-decreasing trend in leaves of different parts of kidney bean plant treated with H₂O and Ca Cl₂;the JA content in middle leaves of kidney bean plant treated with Ca Cl₂ was significantly higher than that of the leaves of the same parts of control plants at 24 h and 48 h,and the upper and lower leaves significantly increased at 48 h.The SA content in the middle,upper and lower leaves of kidney bean plant treated with Ca Cl₂ was significantly higher than that in the same parts of control leaves at 12 h,6 h and 6 h,respectively.（3）When plants were not exposed to WFT-infested,the activities of LOX,AOS,PAL and PR-2 in the upper,middle and lower leaves of kidney bean plant treated with Ca Cl₂ were not significantly different from control plant.When subjected to WFT infestation,the LOX,AOS and PAL activities in middle leaves were much higher in Ca-treated plants than control plant.There was no significant difference in PR-2 activity,but it was higher than that of control plant.LOX activity in upper leaves significantly increased,but LOX activity in lower leaves was not significantly different.The activities of AOS,PAL and PR-2 in upper and lower leaves were not significantly different than control plants.These results indicated that exogenous calcium could induce the systemic resistance of kidney bean plants to WFT.3 Transcriptional analysis of kidney bean plants induced by exogenous Ca in response to WFT infestationTo further study the mechanism of exogenous Ca-induced resistance of kidney bean plants to WFT,high-throughput sequencing technology was used to analyze the transcriptomes of kidney bean plant treated with Ca Cl₂ under WFT infestation.The results showed that a total of 48 928 genes were obtained in the transcriptomic sequence of four samples.Comparison rate involve in reference genome and reference genes were more than 70%and more than 50%,respectively.The numbers of differentially expressed genes in kidney bean plant treated by H₂O and Ca Cl₂ under thrips infestation were 2 403.These differentially expressed genes were categorized into biological process,cell component and molecular function in GO database.In the KEGG database,cellular processes and metabolism were annotated,and most genes performed metabolism.Additionally,we also obtained secondary biomass biosynthesis,plant-pathogen interaction and plant hormone signal transduction in the KEGG database.Furthermore,the results showed that exogenous Ca improved the expression of cyclic nucleotide-gated ion channel（CNGC）genes in kidney bean plant,thereby up-regulated defense genes in four Ca²⁺signaling pathway.Application of exogenous Ca also enhanced the expression of genes involved in linolenic acid pathway and phenylpropane metabolism or isochorismate pathway in kidney bean plants involve in LOX,AOS,PAL and PR-2 genes,promoting the transduction of JA and SA signals.In addition,the secondary biosynthesis genes significantly up-regulated in kidney bean plants treated by Ca Cl₂ were further determined,and a total of 18 flavonoid synthesis genes involving in phenylpropane pathway were detected.There were also 16 genes involving in linolenic acid pathway,including 11 terpenoid synthesis genes,4 alkaloid synthesis genes and 1 glucosinolate biosynthesis gene.The results further revealed that the JA and SA signal transduction pathways play an important regulatory role in kidney bean plants induced by Ca Cl₂ to WFT.4 Counter-defense of WFT to Ca-treated kidney bean plantThe response of the activities of protective enzymes and detoxification enzymes in WFT feeding on control and Ca Cl₂ treated kidney bean plant were investigated in this study.（1）The activities of CAT and SOD in WFT feeding on Ca pre-treated plant were significantly higher than those of control plants,increased by 47.76%and 31.32%,respectively.While POD activity was significantly lower,decreased by 4.61 times.（2）The activities of GST and P450 in WFT feeding on Ca pre-treated plant were significantly higher than those of control plants,increased by 45.87%and 25.24%,respectively.While the activity of Car E significantly decreased by 43.93%.It revealed that WFT enhanced the adaptability to Ca-treated bean plants by changing the activities of protective enzymes and detoxification enzymes.The response of the activities of protective enzymes and detoxification enzymes in WFT to different parts of leaves under Ca-treated the middle leaf of kidney bean plants were also investigated.After Ca Cl₂ treatment the middle leaves of kidney bean plant,（1）the activities of CAT,SOD and POD in WFT feeding on the middle leaves of Ca-treated kidney bean plants were higher than those fed on the upper and lower leaves.The activities of CAT and POD in WFT feeding on the upper,middle and lower leaves of kidney bean plant treated with Ca Cl₂ were significantly higher than those feeding on control plant.However,the SOD activity only significantly increased in the middle leaves.（2）The activities of Car E in WFT feeding on the middle leaves of Ca-treated kidney bean plants were higher than those feeding on the upper and lower leaves,and there was no obvious change in the activities of GST and P450.The activities of GST and P450 of WFT feeding on the upper,middle and lower leaves of kidney bean plant treated with Ca Cl₂ were significantly greater than those feeding on control plants.While the activities of Car E of WFT were only significantly elevated in the middle and lower leaves.The results revealed that WFT increased the activities of protective enzymes and detoxification enzymes to adapt to the enhancement of Ca-induced systemic defense of kidney bean plants.5 Transcriptome analysis of WFT in response to Ca-treated kidney bean plantIn order to reveal the adaptive mechanism of WFT to Ca-induced kidney bean plants,high-throughput sequencing technology was used for transcriptome sequencing to analyze WFT when feeding on control and Ca-treated kidney bean plants.Correlative metabolic pathway was analyzed,and key genes adapted to Ca-treated plants were screened.The results showed that a total of 31 339 genes were sequenced in the transcriptomic sequence of three samples,and the most genes annotated in the NR database were 15 702 genes.There were 1 070 genes differentially expressed genes in WFT when feeding on control and Ca-treated kidney bean plant.These differentially expressed genes were categorized into biological process,cell component and molecular function in GO database,and most genes performed biological processes and cellular components.KEGG metabolic pathway analysis showed that these genes were involved in ribosomes,fatty acid metabolism,lifespan regulation,MAPK signaling and m TOR signaling.The anti-defense genes of WFT against Ca-treated kidney bean plant were further screened.These genes involved in protective enzyme genes,including 2POD genes and 1 SOD gene;involved in detoxification enzyme genes,including 4Car E genes,3 GST genes and 6 P450 genes.There are 19 digestive enzyme genes,including 12 trypsin genes,2 chymotrypsin genes,1 amylase gene,2 cathepsin genes and 2 metalloproteinase genes.In addition,saliva protein genes were also obtained,including 1 Glucosidase gene,2 Alkaline phosphatase gene,1 endo-β-1,4-glucanase（Nl EG1）gene,2 Laccase genes and 1 Glucose oxidase gene,all of which might be involved in adaptation of WFT to Ca-induced kidney bean plant.